CN103735271A - Method for conducting fingerprint identification and analyzed object detection simultaneously through dark-field microscope - Google Patents
Method for conducting fingerprint identification and analyzed object detection simultaneously through dark-field microscope Download PDFInfo
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- CN103735271A CN103735271A CN201310633293.2A CN201310633293A CN103735271A CN 103735271 A CN103735271 A CN 103735271A CN 201310633293 A CN201310633293 A CN 201310633293A CN 103735271 A CN103735271 A CN 103735271A
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Abstract
The invention relates to a method for conducting fingerprint identification and analyzed object detection simultaneously through a dark-field microscope. The method comprises the following steps that a nanometer plasmon probe is made of a nanometer plasmon material and a nucleic acid aptamer; a substrate is processed and a latent fingerprint is obtained through the substrate; the nanometer plasmon probe is dropped to the substrate with the obtained latent fingerprint, so that the latent fingerprint is combined with the nanometer plasmon probe; the dark-field microscope is used for detection. According to the method for conducting fingerprint identification and analyzed object detection simultaneously through the dark-field microscope, the latent fingerprint can be imaged and chemical analyzed objects carried in the fingerprint can be detected simultaneously. A result shows that the method is simple and rapid in operation and can directly conduct nondestructive analysis on a fingerprint sample.
Description
Technical field
The present invention relates to fingerprint analysis detection technique field, be specifically related to utilize dark field microscope to carry out the method for fingerprint recognition and detection of analytes simultaneously.
Background technology
Fingerprint analysis technology has been widely used in individual identification and legal medical expert and has investigated etc. in field.But up to now, in actual applications, due to laten fingerprints be difficult for seen by the naked eye, but very important, the effective evidence staying in scene of a crime, thereby significant in legal medical expert's investigation.But up to the present, fingerprint analysis technology is only confined to confirm this link of personal identification with fingerprint characteristic.And condition is harsh due to the handling procedure complexity to laten fingerprints, required instrument is bulky and be worth valuablely, and in direct-detection people's laten fingerprints, the chemical analyte such as entrained explosive, narcotics also rests on the laboratory research stage.Therefore, develop a kind of simply, fast, directly, harmless, portable, cheap method, can carry out fingerprint imaging simultaneously and detect entrained analyte in fingerprint, seem particularly important.
The method of laten fingerprints imaging is now numerous, grow up at present and mainly comprised by the fingerprint imaging technology that legal medical expert's research field adopted: optical imagery method, powder method, ninhydrin method, iodine fumigation, 502 glue fumigation and vacuum metal sedimentation etc., they are applicable to respectively different fingerprint properties and testing conditions, and respectively have shortcoming.
Optical detection need not be processed fingerprint substantially, can not cause the destruction of fingerprint, is the first-selection in several different methods.But the method is limited in one's ability to the enhancing of developing latent finger prints using, and required instrument and equipment is comparatively huge and complex structure is expensive, cannot extensively popularize, and is not suitable for Site Detection.Powder method is because of the comparatively a kind of process for show of maturation that becomes simple to operate, with low cost, applied widely, but easily fingerprint ridge is damaged, and powder particle is suspended in air, easily enters respiratory tract worker is caused to larger toxic and side effects.Ninhydrin method is simple to operate, and can manifest preferably the laten fingerprints of paper surface, is also the method that evaluation worker extensively adopts at present.But it is poor that the method manifests effect when amino acid content is less in fingerprint, need further subsequent treatment.Iodine fumigation is a kind of harmless process for show, can repeatedly manifest, and is especially applicable to oils laten fingerprints.But the light background colour that is subject to of the method colour developing disturbs, the difficulty while extracting of taking pictures.And iodine easily distils, human body is also had to certain toxic action.The 502 glue methods of exerting a gradual, corrupting influence on are to process the effective ways of non-porous lip-deep laten fingerprints.But it is a problem that the method is processed the contrast of rear fingerprint and background, so also need some post processings.Vacuum metal sedimentation is to process the another kind of important technology of non-porous lip-deep laten fingerprints.But instrument is comparatively expensive, and complex operation, brings very large obstacle to Site Detection.
Outside the technology of commercialization extensive use, the technology growing up in recent years also has: fluorescence method, with rare earth material, quantum dot or fluorescent dye, be attached on fingerprint, and by fluorescence microscope, but material requested is to the toxic effect of operator.Electrochemical process, mainly contains scan-type electrochemical microscope method, Electrochemiluminescince and electrochemistry-surface plasma body resonant vibration method, and some method can detect the analyte in fingerprint, but needs valuable large-scale instrument to realize.Mass spectrography, can detect in fingerprint the analyte of trace, but fingerprint sample need to be dissolved or extract, and being a kind ofly has complete destructive method to fingerprint, and needs large-scale mass spectrograph.By Vibrational Spectrometry, mainly refers to FT-IR & FT-RAMAN spectra, also can detect the analyte in fingerprint, and shortcoming is also to purchase large-scale instrument, and needs long-time operation.At present these methods have and much can accomplish to detect the analyte carrying in fingerprint, but weak tendency separately also clearly.
In a word, in the existing technology having been adopted by legal medical expert field, there is respectively its shortcoming, and the most important thing is outside imaging, to detect chemical analyte wherein; Some can detect analyte the advanced technology developing in recent years, but can not realize nondestructive analysis, and needs complicated valuable large-scale instrument, is unfavorable for extensively popularizing and Site Detection; In addition, by dark field microscope, research and the application for fingerprint analysis field have not been reported.
Summary of the invention
The object of this invention is to provide a kind of dark field microscope that utilizes and carry out the method for fingerprint recognition and detection of analytes simultaneously, when first dark field microscope being applied to fingerprint analysis, chemical analyte is detected.
The dark field microscope that utilizes of the present invention carries out the method for fingerprint recognition and detection of analytes simultaneously, comprises the steps: S1, utilizes nano plasma excimer material and aptamer to prepare nano plasma excimer probe; S2, processes substrate and utilizes this substrate to obtain laten fingerprints; S3, drips described nano plasma excimer probe in obtaining in the substrate of laten fingerprints, and described laten fingerprints is combined with described nano plasma excimer probe; S4, utilizes dark field microscope to detect.The invention provides a kind of novel nano plasma excimer probe based on aptamer under dark field microscope to laten fingerprints imaging, and detect the universal method of the chemical analyte carrying in fingerprint simultaneously.Wherein, aptamer refers to the energy specific bond protein that goes out through in-vitro screening technology screening or the oligonucleotide fragment of other small-molecule substances, is the general name of a class single stranded nucleic acid molecule.Nano plasma excimer probe refers to the probe of making at nano level plasmon material by size.
Preferably, described nano plasma excimer material is nanometer gold.Because nanometer gold itself has good biocompatibility, make nano plasma excimer probe provided by the present invention there is good biocompatibility, thereby make probe provided by the present invention to operator without any toxic action.Wherein, so-called plasmon is conventional conception, refers to the mode of electromagnetic wave that light and nano metal surface electronic interact and cause, the nano material with this character is called nano plasma excimer material.The preparation of nanometer gold can be carried out according to known method of the prior art.
Preferably, described nanometer gold is the spherical gold nano grain of diameter 40～60nm, or the cube shaped gold nano grain of the length of side 40～60nm.In fact, this nanometer gold can be the gold nano grain of any size, shape, and the numerical value of above-mentioned 40～60nm only illustrates as preferred embodiment.
Preferably, described aptamer is the aptamer of cocaine, lysozyme, adenosine triphosphate, Tianning, morphine or cannabinol.Should be appreciated that, method provided by the invention has universality, only need to first select and the corresponding aptamer of analyte according to spirit of the present invention.When described aptamer is cocaine aptamer, the lowest detectable limit of cocaine is preferably at about 1.5～2cm
2fingerprint area on 90ng cocaine.
Preferably, described step S1 comprises and first aptamer is cut into two single stranded DNAs, thereby makes described nano plasma excimer probe have two types.Adopt the reason of the probe of two types to be: when there is no analyte, this probe of two types can not combine, and is the dispersity of individual particle, aobvious green under the microscope; When there is analyte, this probe of two types can combine, and is the coherent condition of several granules, under the microscope aobvious orange or red.More preferably, 3 ' of these two single stranded DNAs end or 5 ' end add respectively some T bases, for example 10.Meanwhile, these two single stranded DNAs are respectively by 3 ' end sulfydryl modification or 5 ' end sulfydryl modification.
Preferably, on every kind of described nano plasma excimer probe, be combined with the irrelevant sequence of not being combined with described laten fingerprints.When two single stranded DNAs are assembled into respectively on nano plasma excimer material, also comprise the single stranded DNA of an irrelevant sequence is assembled on nano plasma excimer material simultaneously.Theoretically, base number is less than any single stranded DNA of 10 can be used as irrelevant sequence DNA, and the effect of this irrelevant sequence DNA is the packing density of rationally controlling these two DNA.What on the nano plasma excimer probe of two types, assemble is respectively a DNA single chain and this irrelevant sequence and another DNA single chain and this irrelevant sequence.When 3 ' end of single stranded DNA or 5 ' end are while being added with some T bases sulfydryl modification, this irrelevant sequence be the DNA of these some T base sulfydryl modifications, and for example this irrelevant sequence is 3 ' to hold 10 T bases of sulfydryl modification.
Preferably, described substrate is microscope slide or the indium tin oxide-coated glass sheet of observing for dark field microscope.In fact, one of reason of using indium tin oxide-coated glass sheet in the present invention is in order to confirm the gathering of gold nano grain for sem observation.Further, this substrate can also be the material of the flat transparent such as rigid plastic sheet, lucite.
Preferably, described step S2 comprises sealer is covered in the substrate of obtaining laten fingerprints.Sealer solution is dripped to surface of glass slide, so sealer can wrap up surface of glass slide completely, probe or be adsorbed on fingerprint, or just directly contact with sealer, and can not contact with slide.Probe contacts with sealer, can not be adsorbed onto on sealer.If probe contacts with slide, can be adsorbed onto on slide, cannot distinguish with the probe being adsorbed onto on fingerprint, just cannot imaging.This sealer can reduce background, improves signal to noise ratio, thereby improves sensitivity and the specificity of imaging and detection.In described step S2, the processing of substrate is comprised and utilizes suds, acetone, ethanol and deionized water to clean substrate, to remove dust and the Organic substance of surface of glass slide, and other influences is observed and the impurity of imaging effect.
Preferably, described sealer is to contain and the protein solution of described nano plasma excimer probe without any non-specific binding.
Preferably, described sealer is casein solution.The concentration of this casein solution is 0.1%～2%(w/v preferably, mass volume ratio), be more preferably 1%.
Preferably, described step S3 comprises and utilizes phosphate buffer and deionized water rinsing, to remove unconjugated described nano plasma excimer probe.Described step S3 is also included in substrate two kinds of probes that drip with concentration.Preferably, the concentration of two kinds of probes is preferably and is 0.1～0.5nM, more preferably 0.2nM.
Preferably, described step S4 comprises and utilizes inverted microscope fit on dark field condenser to observe.More preferably, dark field microscope of the present invention is the object lens that adopt inverted microscope fit on dark field condenser (scope of its numerical aperture (NA) is 0.8<NA<0.95) and different amplification (60 times and 4 times (NA=0.8)), and adopt Halogen light (100W) as white light source, adopting colour charge coupling element (CCD) is image-forming component, be transferred on computer and show, and with software control be processed into picture.
In fact, at present also without any utilizing dark field microscope to carry out the report of fingerprint imaging, dark field microscope is generally used for observing Brownian movement.Wherein it should be noted in the discussion above that the thing that is observed due to Brownian movement is dynamic, and the object of the invention is fingerprint to carry out imaging, this just requires to be observed thing and can not arbitrarily move.The present invention utilizes analyte in fingerprint and the interaction between nano plasma excimer probe (having modified the gold nano grain of the aptamer with specific recognition function), probe is fixed in substrate, thereby reaches the object of imaging and detection.In addition, consider that probe and substrate divide other charge property, nonspecific Electrostatic Absorption is inevitable, and this can have a strong impact on imaging effect and detection sensitivity and specificity.So this is the difficult point that cannot avoid in operating process of the present invention, method provided by the invention---at substrate surface, cover one deck sealer layer and successfully solved this difficult problem.Finally, than other, the advantage by the laten fingerprints formation method of the luminescence generated by light of fluorescence microscope is that the luminous intensity of probe is high and optical property stable in the present invention, and operator is not had to toxic action.
Fingerprint analysis method provided by the invention not only can be carried out imaging to laten fingerprints, and can detect chemical analyte entrained in fingerprint simultaneously.Result shows, the present invention is to provide a kind of simple and quick method, can carry out direct, harmless analysis to fingerprint sample.In the present invention, the main gold nano grain using has good biocompatibility, and other required chemistry, biomaterial are also all without human body toxic.Reagent chemicals required for the present invention and instrument and equipment are relatively inexpensive portable, are easy to standardization, commercialization and large-scale popularization.Therefore the imaging that the present invention is laten fingerprints and detection and the practical application thereof of analyte provide a kind of new thinking and technical support.
Accompanying drawing explanation
Fig. 1 is the low range dark field microscope photo result of laten fingerprints imaging in embodiment 1;
Fig. 2 is the qualitative results of fingerprint imaging while observing with high magnification dark field microscope the cocaine that carries different quality in fingerprint in embodiment 1;
Fig. 3 is that in embodiment 1, high magnification dark field microscope is observed fingerprint imaging luminous point to carry the result of different quality cocaine in half-quantitative detection fingerprint;
Fig. 4 is the qualitative results of fingerprint imaging while observing the specific detection of carrying cocaine in fingerprint with high magnification dark field microscope in embodiment 1;
Fig. 5 is the semi-quantitative results of fingerprint imaging luminous point while observing the specific detection of carrying cocaine in fingerprint with high magnification dark field microscope in embodiment 1.
The specific embodiment
Below in conjunction with accompanying drawing, provide preferred embodiment of the present invention, and be described in detail.
Embodiment 1: the cocaine that the nano plasma excimer probe based on aptamer carries in the laten fingerprints imaging on microscope slide also being detected to fingerprint simultaneously under dark field microscope
(1) preparation of nano plasma excimer probe: 150mL trisodium citrate aqueous solution (2.2mM) is heated to the 15min that boils, adds 1mL aqueous solution of chloraurate (25mM) reaction 15min.Be cooled to 95 ℃, add 1mL aqueous solution of chloraurate (25mM) reaction 30min, repeat twice.Then take out 55mL sample, and add 53mL water and 2mL trisodium citrate aqueous solution (60mM).Repeat the step 6 time that adds 1mL aqueous solution of chloraurate (25mM) reaction to grow up, the spherical gold nano grain Au NPs(the method that obtains diameter 50nm can also be prepared the spherical gold nano grain of diameter 40～60nm, only needs to change final step and adds 1mL aqueous solution of chloraurate (25mM) to react the number of repetition of the step of growing up).By the concentration of 5 μ L, be the single stranded DNA 1(ss-DNA1 of 100 μ M, sequence is 5 '-GTTCTTCAATGAAGTGGGACGACATTTTTTTTTT-SH-3 ') or single stranded DNA 2(ss-DNA2, sequence is 5 '-GGGAGTCAAGAACTTTTTTTTTT-SH-3 ') with the concentration of 5 μ L be the single stranded DNA 3(ss-DNA3 of 100 μ M, sequence is 5 '-TTTTTTTTTT-SH-3 ') (wherein ss-DNA1 and ss-DNA2 are the aptamer of cocaine two DNA after cut to join incubated at room in the above-mentioned gold nano grain solution (0.2nM) of 400 μ L, ss-DNA3 is the DNA of an irrelevant sequence).Add phosphate buffer (PB) to 10mM, sodium chloride (NaCl) solution is to 0.1M, and pH7.0, hatches 40 hours.At 4 ℃, leave heart 20min in 12000, supernatant discarded, is scattered in 400 μ L sodium ascorbyl phosphate buffer (PBS) after washing 3 times.
(2) obtaining of the processing of fingerprint substrate and laten fingerprints: the microscope slide of dark field microscope is cleaned to 30min, N in suds, acetone, ethanol, deionized water for ultrasonic respectively
2dry up.Slide seals with casein, by 200 μ L be dissolved in casein in PBS (1%, w/v) be added drop-wise on slide, at 37 ℃, hatches 2 hours, and at 4 ℃, hatch 12 hours, and after washing dries up.The cocaine of finger wash clean back loading different quality is then pressed collection fingerprint lightly on slide.
(3) finger prints processing process: on slide, drip respectively two kinds of different nano plasma excimer probes of 45 μ L (Au NPs – DNA1 and Au NPs – DNA2, the concentration of two kinds of probes is 0.2nM,, under room temperature, hatch 30min.With PBS and deionized water rinsing and dry up, to remove unconjugated probe.
(4) dark field microscope detects: the object lens (NA=0.8) of inverted microscope fit on dark field condenser (scope of its numerical aperture (NA) is 0.8<NA<0.95) and 60 times and 4 times, white light source is the Halogen light of 100W, image-forming component is colored CCD, and switching is shown on computer.
Adopt the details in a play not acted out on stage, but told through dialogues photochrome of 4 times of object lens observation laten fingerprintses on dark field microscope to the results are shown in accompanying drawing 1.In fact, due to the details in a play not acted out on stage, but told through dialogues visual field that the size of fingerprint produces much larger than condenser lens, this photo comes from the result of the details in a play not acted out on stage, but told through dialogues photochrome splicing of multiple different finger-print regions.This figure shows to contrast between bright fingerprint projection and dark substrate, and this result provides a clear and legible fingerprint profile.And except the style (resolution of the first level of fingerprint) of fingerprint projection, can also clearly identify fingerprint the second level (comprising end, intersection, Shuan Chahe island etc.) and tri-layer (pore).This shows that method provided by the invention can provide clear-cut, resolution is high, contrast is high imaging results.Wherein, red circle represents fingerprint the second level (comprising end, intersection, Shuan Chahe island etc.), and green circle represents fingerprint tri-layer (pore).
The fingerprint of cocaine that load has a different quality under high-resolution dark field microscope observable details in a play not acted out on stage, but told through dialogues photochrome as accompanying drawing 2.From a to h, the load capacity of cocaine is respectively 0ng, 150ng, 300ng, 750ng, 1.5 μ g, 3 μ g, 15 μ g and 30 μ g.Result shows that high-definition picture changes to redness from green on the whole gradually along with the increase of the load capacity of cocaine, that is to say that green luminous point reduces gradually and orange-red luminous point increases gradually.Our verified orange, red luminous point comes from the gold nano grain of cocaine induction and assembles, therefore, we have chosen 300 luminous points in the region of fingerprint projection at random, has added up the wherein orange and red shared percentage ratio of luminous point, the results are shown in accompanying drawing 3.Can see between this percentage ratio and the logarithm of cocaine quality and have good linear relationship.Calculate (signal value of blank sample adds that the standard deviation of the blank sample of 3 times is lowest detectable limit) and show that the lowest detection of cocaine load capacity is limited to 90ng.
Adopt this kind of analytical method to detect two of cocaine kinds of metabolite ecgonine benzoate .s (benzoyl ecgonine, BE) and ecgonine methyl ester (ecgonine methyl ester, EME).These two kinds of materials and cocaine chemistry structure is very similar, and we using this proof of analog as cocaine cocaine detection specificity.Load has the cocaine of equal in quality and the fingerprint of analog thereof observable details in a play not acted out on stage, but told through dialogues photochrome under high-resolution dark field microscope as accompanying drawing 4, to be respectively blank group, BE group, EME group and cocaine group from a to d.The specific detection result obtaining with our semi-quantitative method is as accompanying drawing 5.Between the result that the result that two kinds of analog obtain and cocaine obtain, there is huge difference, be almost equivalent to dummy.Above result shows, all shows better detection performance aspect the susceptiveness of the cocaine that the nano plasma excimer probe based on aptamer in the present invention carries detect fingerprint under dark field microscope in and specificity.
Embodiment 2: the cocaine that the nano plasma excimer probe based on aptamer carries in the laten fingerprints imaging on microscope slide also being detected to fingerprint simultaneously under dark field microscope
(1) preparation of nano plasma excimer probe: gold chloride is joined in the hexadecyltrimethylammonium chloride aqueous solution (0.1M) of 10mL, to its concentration be 0.25mM.The sodium borohydride aqueous solution (20mM) that adds 0.45mL, 30 ℃ are reacted 1 hour, as seed solution.Configure two bottles of growth-promoting medias: 320mg hexadecyltrimethylammonium chloride is added in 9.605mL deionized water, add successively 0.25mL aqueous solution of chloraurate (10mM), 0.01mL aqueous sodium bromide (10mM), 0.09mL aqueous ascorbic acid (40mM).Reaction 30min, repeats twice.Then take out 55mL sample, and add 53mL water and 2mL trisodium citrate aqueous solution (60mM).In one bottle of growth-promoting media, add the above-mentioned seed solution of 0.45mL and vibrate 5 seconds, then therefrom taking out 0.45mL and proceed to another bottle of growth-promoting media.Fully mix 10 seconds, in 30 ℃ of reactions 15 minutes, centrifugal concentrating, to 1mL, obtains the cube shaped gold nano grain (Au NPs) (the method can also be prepared the cube shaped gold nano grain of diameter 40～60nm, only needs to change the volume that final step adds seed solution) of length of side 45nm.By the concentration of 5 μ L, be the single stranded DNA 1(ss-DNA1 of 100 μ M, sequence is 5 '-GTTCTTCAATGAAGTGGGACGACATTTTTTTTTT-SH-3 ') or single stranded DNA 2(ss-DNA2, sequence is 5 '-GGGAGTCAAGAACTTTTTTTTTT-SH-3 ') with the concentration of 5 μ L be the single stranded DNA 3(ss-DNA3 of 100 μ M, sequence is 5 '-TTTTTTTTTT-SH-3 ') (wherein ss-DNA1 and ss-DNA2 are that the aptamer of cocaine is by two DNA after reasonably cutting to join incubated at room in the above-mentioned gold nano grain solution of 400 μ L, ss-DNA3 is the DNA of an irrelevant sequence).Add phosphate buffer (PB) to 10mM, sodium chloride (NaCl) solution is to 0.1M, and pH7.0, hatches 40 hours.At 4 ℃, leave heart 20min in 12000, supernatant discarded, is scattered in 400 μ L sodium ascorbyl phosphate buffer (PBS) after washing 3 times.
(2) obtaining of the processing of fingerprint substrate and laten fingerprints: the microscope slide of dark field microscope is cleaned to 30min, N in suds, acetone, ethanol, deionized water for ultrasonic respectively
2dry up.Slide seals with casein, by 200 μ L be dissolved in casein in PBS (1%, w/v) be added drop-wise on slide, at 37 ℃, hatches 2 hours, and at 4 ℃, hatch 12 hours, and after washing dries up.The cocaine of finger wash clean back loading different quality is then pressed collection fingerprint lightly on slide.
(3) finger prints processing process: drip respectively two kinds of different nano plasma excimer probes of 45 μ L (Au NPs – DNA1 and Au NPs – DNA2, concentration is 0.2nM, preferable range is 0.1～0.5nM) on slide, hatch 30min under room temperature.With PBS and deionized water rinsing and dry up.
(4) dark field microscope detects: the object lens (NA=0.8) of inverted microscope fit on dark field condenser (scope of its numerical aperture (NA) is 0.8<NA<0.95) and 60 times and 4 times, white light source is the Halogen light of 100W, image-forming component is colored CCD, and switching is shown on computer.
Embodiment 3: the adenosine triphosphate that the nano plasma excimer probe based on aptamer carries in the laten fingerprints imaging on microscope slide also being detected to fingerprint simultaneously under dark field microscope
(1) preparation of nano plasma excimer probe: 150mL trisodium citrate aqueous solution (2.2mM) is heated to the 15min that boils, adds 1mL aqueous solution of chloraurate (25mM) reaction 15min.Be cooled to 95 ℃, add 1mL aqueous solution of chloraurate (25mM) reaction 30min, repeat twice.Then take out 55mL sample, and add 53mL water and 2mL trisodium citrate aqueous solution (60mM).Repeat the step 6 time that adds 1mL aqueous solution of chloraurate (25mM) reaction to grow up, obtain the spherical gold nano grain (Au NPs) (the method can also be prepared the spherical gold nano grain of diameter 40～60nm, only needs to change final step and adds 1mL aqueous solution of chloraurate (25mM) to react the number of repetition of the step of growing up) of diameter 50nm.By the concentration of 5 μ L be 100 μ M single stranded DNA 1 ' (ss-DNA1 ', sequence is 5 '-HS-TTTTTTTTTTACCTGGGGGAGTAT-3 ') or single stranded DNA 2 ' (ss-DNA2 ', sequence is 5 '-HS-TTTTTTTTTTTGCGGAGGAAGGT-3 ') with the concentration of 5 μ L be 100 μ M single stranded DNA 3 ' (ss-DNA3 ', sequence is 5 '-TTTTTTTTTT-SH-3 ') (wherein ss-DNA1 ' and ss-DNA2 ' they are that the aptamer of adenosine triphosphate is by two DNA after reasonably cutting to join incubated at room in the above-mentioned gold nano grain solution (0.2nM) of 400 μ L, ss-DNA3 ' is the DNA of an irrelevant sequence).Add phosphate buffer (PB) to 10mM, sodium chloride (NaCl) solution is to 0.1M, and pH7.0, hatches 40 hours.At 4 ℃, leave heart 20min in 12000, supernatant discarded, is scattered in 400 μ L sodium ascorbyl phosphate buffer (PBS) after washing 3 times.
(2) obtaining of the processing of fingerprint substrate and laten fingerprints: the microscope slide of dark field microscope is cleaned to 30min, N in suds, acetone, ethanol, deionized water for ultrasonic respectively
2dry up.Slide seals with casein, by 200 μ L be dissolved in casein in PBS (1%, w/v) be added drop-wise on slide, at 37 ℃, hatches 2 hours, and at 4 ℃, hatch 12 hours, and after washing dries up.The adenosine triphosphate of finger wash clean back loading different quality is then pressed collection fingerprint lightly on slide.
(3) finger prints processing process: drip respectively two kinds of different nano plasma excimer probes of 45 μ L (Au NPs – DNA1 and Au NPs – DNA2, concentration is 0.2nM, preferable range is 0.1～0.5nM) on slide, hatch 30min under room temperature.With PBS and deionized water rinsing and dry up.
(4) dark field microscope detects: the object lens (NA=0.8) of inverted microscope fit on dark field condenser (scope of its numerical aperture (NA) is 0.8<NA<0.95) and 60 times and 4 times, white light source is the Halogen light of 100W, image-forming component is colored CCD, and switching is shown on computer.
Embodiment 4: the cocaine that the nano plasma excimer probe based on aptamer carries in the laten fingerprints imaging on indium tin oxide-coated glass also being detected to fingerprint simultaneously under dark field microscope
(1) preparation of nano plasma excimer probe: 150mL trisodium citrate aqueous solution (2.2mM) is heated to the 15min that boils, adds 1mL aqueous solution of chloraurate (25mM) reaction 15min.Be cooled to 95 ℃, add 1mL aqueous solution of chloraurate (25mM) reaction 30min, repeat twice.Then take out 55mL sample, and add 53mL water and 2mL trisodium citrate aqueous solution (60mM).Repeat the step 6 time that adds 1mL aqueous solution of chloraurate (25mM) reaction to grow up, obtain the spherical gold nano grain (Au NPs) (the method can also be prepared the spherical gold nano grain of diameter 40～60nm, only needs to change final step and adds 1mL aqueous solution of chloraurate (25mM) to react the number of repetition of the step of growing up) of diameter 50nm.By the concentration of 5 μ L, be the single stranded DNA 1(ss-DNA1 of 100 μ M, sequence is 5 '-GTTCTTCAATGAAGTGGGACGACATTTTTTTTTT-SH-3 ') or single stranded DNA 2(ss-DNA2, sequence is 5 '-GGGAGTCAAGAACTTTTTTTTTT-SH-3 ') with the concentration of 5 μ L be the single stranded DNA 3(ss-DNA3 of 100 μ M, sequence is 5 '-TTTTTTTTTT-SH-3 ') (wherein ss-DNA1 and ss-DNA2 are that the aptamer of cocaine is by two DNA after reasonably cutting to join incubated at room in the above-mentioned gold nano grain solution of 400 μ L, ss-DNA3 is the DNA of an irrelevant sequence).Add phosphate buffer (PB) to 10mM, sodium chloride (NaCl) solution is to 0.1M, and pH7.0, hatches 40 hours.At 4 ℃, leave heart 20min in 12000, supernatant discarded, is scattered in 400 μ L sodium ascorbyl phosphate buffer (PBS) after washing 3 times.
(2) obtaining of the processing of fingerprint substrate and laten fingerprints: indium tin oxide-coated glass sheet is cleaned to 30min, N in suds, acetone, ethanol, deionized water for ultrasonic respectively
2dry up.Slide seals with casein, by 200 μ L be dissolved in casein in PBS (1%, w/v) be added drop-wise on slide, at 37 ℃, hatches 2 hours, and at 4 ℃, hatch 12 hours, and after washing dries up.The cocaine of finger wash clean back loading different quality is then pressed collection fingerprint lightly on slide.
(3) finger prints processing process: drip respectively two kinds of different nano plasma excimer probes of 45 μ L (Au NPs – DNA1 and Au NPs – DNA2, concentration is 0.2nM, preferable range is 0.1～0.5nM) on slide, hatch 30min under room temperature.With PBS and deionized water rinsing and dry up.
(4) dark field microscope detects: the object lens (NA=0.8) of inverted microscope fit on dark field condenser (scope of its numerical aperture (NA) is 0.8<NA<0.95) and 60 times and 4 times, white light source is the Halogen light of 100W, image-forming component is colored CCD, and switching is shown on computer.
Above-described, be only preferred embodiment of the present invention, not in order to limit scope of the present invention, the above embodiment of the present invention can also make a variety of changes.Be that simple, the equivalence that every claims according to the present patent application and description are done changes and modify, all fall into the claim protection domain of patent of the present invention.The present invention not detailed description be routine techniques content.
Claims (12)
1. utilize dark field microscope to carry out the method for fingerprint recognition and detection of analytes simultaneously, it is characterized in that, described method comprises the steps:
S1, utilizes nano plasma excimer material and aptamer to prepare nano plasma excimer probe;
S2, processes substrate and utilizes this substrate to obtain laten fingerprints;
S3, drips described nano plasma excimer probe in obtaining in the substrate of laten fingerprints, and described laten fingerprints is combined with described nano plasma excimer probe;
S4, utilizes dark field microscope to detect.
2. method according to claim 1, is characterized in that, described nano plasma excimer material is nanometer gold.
3. method according to claim 2, is characterized in that, described nanometer gold is the spherical gold nano grain of diameter 40～60nm, or the cube shaped gold nano grain of the length of side 40～60nm.
4. method according to claim 1, is characterized in that, described aptamer is the aptamer of cocaine, lysozyme, adenosine triphosphate, Tianning, morphine or cannabinol.
5. method according to claim 1, is characterized in that, described step S1 comprises and first aptamer is cut into two single stranded DNAs, thereby makes described nano plasma excimer probe have two types.
6. method according to claim 5, is characterized in that, on every kind of described nano plasma excimer probe, is combined with the irrelevant sequence of not being combined with described laten fingerprints.
7. method according to claim 1, is characterized in that, described substrate is microscope slide or the indium tin oxide-coated glass sheet of observing for dark field microscope.
8. method according to claim 1, is characterized in that, described step S2 comprises sealer is covered in the substrate of obtaining laten fingerprints.
9. method according to claim 8, is characterized in that, described sealer is to contain and the protein solution of described nano plasma excimer probe without any non-specific binding.
10. method according to claim 9, is characterized in that, described sealer is casein solution.
11. methods according to claim 1, is characterized in that, described step S3 comprises and utilizes phosphate buffer and deionized water rinsing, to remove unconjugated described nano plasma excimer probe.
12. methods according to claim 1, is characterized in that, described step S4 comprises and utilizes inverted microscope fit on dark field condenser to observe.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106093005A (en) * | 2016-06-06 | 2016-11-09 | 上海海洋大学 | A kind of latent fingerprint high definition recognition methods based on lysozyme Raman spectrum imaging |
| CN106841150A (en) * | 2017-03-17 | 2017-06-13 | 浙江警察学院 | A kind of method that fingerprint leaves various illicit drugs identifications and fingerprint imaging |
| CN113125410A (en) * | 2021-04-19 | 2021-07-16 | 湖南大学 | Multi-material-substrate universal latent fingerprint imaging and residue detection system based on gold-graphite nanocapsule particles and imaging method thereof |
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2013
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| LI KUN 等: "Nanoplasmonic Imaging of Latent Finger-prints and Identification of Cocaine", 《ANGEWANDTE CHEMIE, INTERNATIONAL EDITION》 * |
| R. LEGGETT 等: ""Intelligent" Fingerprinting: Simultaneous Identification of Drug Metabolites and Individuals by Using Antibody-Functionalized Nanoparticles", 《ANGEWANDTE CHEMIE, INTERNATIONAL EDITION》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106093005A (en) * | 2016-06-06 | 2016-11-09 | 上海海洋大学 | A kind of latent fingerprint high definition recognition methods based on lysozyme Raman spectrum imaging |
| CN106841150A (en) * | 2017-03-17 | 2017-06-13 | 浙江警察学院 | A kind of method that fingerprint leaves various illicit drugs identifications and fingerprint imaging |
| CN106841150B (en) * | 2017-03-17 | 2019-10-15 | 浙江警察学院 | A kind of method that fingerprint leaves a variety of illicit drugs identifications and fingerprint imaging |
| CN113125410A (en) * | 2021-04-19 | 2021-07-16 | 湖南大学 | Multi-material-substrate universal latent fingerprint imaging and residue detection system based on gold-graphite nanocapsule particles and imaging method thereof |
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